Animal venoms are complex biochemical cocktails capable of diverse and extremely potent physiological effects. Venoms are also highly prized as novel sources of pharmaceuticals and are essential for the development of lifesaving antivenoms. Despite their scientific and clinical significance, the molecular composition and functional activities of many biomedically important venoms remains poorly known. The long-term objective of this proposal is to understand the molecular complexity, function and evolution of black widow spider venom. Black widow spiders possess a highly neurotoxic venom that causes severe pain in humans. While black widow venom has played a critical role in the study of vertebrate neurotransmission, the molecular composition of this venom and its functional and biomedical applications have not been investigated using modern molecular techniques. The proposed work will address this significant knowledge gap through three specific aims. First, a comprehensive black widow venom gland transcriptome will be assembled using next-generation sequencing methods to characterize the genes expressed in venom gland tissue. Second, the set of transcripts involved in venom function will be identified from the transcriptome. This will involve the proteomic technique of peptide fingerprinting to determine the proteins in venom samples using mass spectrometry and bioinformatic screens of a transcriptome database. The next-generation sequencing application of Tag-Profiling will also be used to identify genes that exhibit venom-specific patterns of expression. The third specific aim will examine the functional evolution of black widow venom through cross-species comparisons of venom gene variation that quantify patterns of selection, as well as comparative analyses of gene expression. Phylogenetic analyses of functionally important toxin proteins will also reveal the molecular mechanisms that have contributed to the extreme toxicity of black widow spider venom. When completed, the proposed work will provide an unparalleled view of the molecular diversity, evolution and biomedical utility of black widow venom. The results of this project will also generate significant public genomic resources and provide a foundation for future studies aimed at understanding the molecular mechanisms of toxin functional evolution. PUBLIC HEALTH RELEVANCE: The major goal of this proposal is to determine the molecular composition, functional activities and evolution of black widow spider venom. The project has significant relevance for public health because black widow venom is extremely hazardous to humans, but it is also an essential tool in the study of vertebrate neurophysiology. The proposed work will provide an unprecedented understanding of the molecular basis for the extreme toxicity of black widow spiders and will aid in the development of novel pharmaceuticals and improved antivenoms.